Hello Andres,

BL21AI won't be of much help as compared with any E. coli host, since it is designed to express T7 RNA polymerase under the control of an arabinose promoter, whereas your gene is under the control of a T5 promoter, which is recognized by E. coli polymerase. My basic questions are : a) did you check that the sequence of your plasmid is correct (ORF in proper reading frame, no point mutations); b) did you check whether your protein might be insoluble; c) did you compare the Coomassie stain of extracts from induced and non-induced cultures : it might be that your protein is subject to proteolysis and generates fragments that are  no longer recognized by your antibody (admittedly, this latter scenario is a bit far-fetched). 

Good luck,

Pierre

BL21 Rosetta strains is very good for the expreesion from pET28a series of vectors. Better to clone in that also and check the same.

A detailed description of factors affecting expression in general is provided in the link:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4029002/

 Hello Andres,

Despite the use of strong transcriptional and translational signals, level of protein expression remains low sometimes. You should optimize conditions for expression again by varying incubation temperature, isopropyl β- D-1-thiogalactopyranoside (IPTG) concentration and incubation time.  What I am interested in is the kind of protein expressed. Are you sure the proteins in questions can be actually expressed in E. coli BL21 AI. Protein you are expressing may be toxic to the bacteria. Do check if there is a slowdown of growth conditions upon induction compared to your control.

Also check whether the codon usage of your target protein differs much from the average codon usage of the expression host you are using, this can possibly create expression problems.

You may try adding ethanol to a finale concentration of 2-3% while inducing expression of your protein using IPTG . Mine worked out this way while working for my master thesis, I tried both with and without ethanol.  Interestingly, I noticed that ethanol produces several fold increase in expression and enhances the solubility. It works with both T5 and T7 promoter. I used this trick long back. 

Although I do not know the exact molecular mechanism behind increased  expression of protein using ethanol. But since ethanol is amphipathic molecule and can influence the cellular environment of the cell by making changes in the membrane e.g. its lipid composition, transport, membrane protein assembly etc. and these changes may further affect the membrane associated events, such as DNA replication. It possibly leads to the enhancement of DNA synthesis, hence gene amplification which can cause the enhancement of inducible protein synthesis in cells.  

Happy to try to discuss this further with you.

All the best.

Hello, everybody.

Thank you for all the time and the answers you have provided me. I would like to provide answers to the questions that you have asked me, I really appreciate your help.

@Pierre Béguin, Thanks for your help, here are the answers to the questions:

1. The sequence of the plasmid seems to be OK. As I have been having issues with the expression, I'm currently working with the DNA that was provided to me directly from the gene synthesis manufacturer so that, at least in theory, shall not be an issue. 

2. For all the detection procedures I've been working with cellular lysates that are being performed in PBS buffer (soluble fraction) and Urea-TRIS buffer (insoluble fractions). In any of the previously mentioned buffers I've been able to detect the protein nor in dot-blot or Western Blot (antibodies are working fine with detection positive controls). Lysates are being produced in FastPrep at 6 m/s for 40 seconds, 5 cycles. The lysis seems to be working effectively as the detection positive control is being obtained properly. Urea-TRIS extraction buffer seems also to be working fine as the positive control is an insoluble protein found only in the insoluble fraction. 

3. Coomasie stains of the induced and non induced cultures (including soluble and insoluble extracts) show no over-expression bands, the profiles seem identical.

If there is any more information I can provide to you please don't hesitate in telling me, I'm sure that any assistance you could provide me will be really helpful.

@Ritu Rathe, Thank you as well. Please find below the answers to your questions:

1. I will try to vary the IPTG concentration for the inductions to see if any positive results come. I will tell you if I achieve any advances.

2. With my team we chose BL21 AI strain as it has a lowered protease activity and seemed capable of toxic protein synthesis, compared to the negative control (empty pUC-57 vector) the growth of the transformed cultures is quite slow and it does not seem to be lowered after the induction. We expect the delayed growth as the gene we're trying to synthesise is about 3000 pb.

3. Codon usage was optimised previous to the gene synthesis so this in our case shall not be a problem. 

4. I've also heard of the add-ethanol technique, I will be experimenting with it after trying some of the "traditional" methods, I think it would be of great help, however at this stage I only aim to obtain the protein under a standardised technique.

@Laurence Stuart Hall. Thank you for the information and links provided, I saw in one of the links that you provide a heat-shock procedure prior to the induction. This procedure could be really useful, I will try it and tell you if there are any positive results. I will also try to titrate the IPTG concentration that is being used for induction because, as you also mentioned, it is indeed a critical point in the expression procedure. 

Thanks to everybody for all your help, if there is any more information that could be of help in solving the issue i'm having, please do not hesitate in telling me.

I really appreciate all the information that you are providing me.

Regards, 

Andrés.